Troubleshooting Chemiluminescent Western Blots: Possible Cause 4 for Weak Signals – Blot Processing

Sometimes life in the lab gets crazy, right? You are finishing a Western blot and you realize that you are supposed to be at an important lecture across campus in 10 min!! Or, your spouse calls to say that one of the kids needs to be picked up as soon as possible. Yikes! The challenge is that blots should be processed and detected on the same day. And, the secondary antibody should be incubated the day of imaging and fresh substrate added just before imaging. Is it that important to your results? Yes, it is and just to prove it, we did a few experiments.

In Table 1, we studied performance differences when the same blot is imaged immediately after processing vs. stored overnight dry and then imaged. In Table 2, we looked at performance differences when the same blot is imaged immediately after processing vs. stored overnight wet and then imaged. Blots in both tables were all imaged on the C-DiGit® Blot Scanner. (And, all images are normalized to the Lookup Tables (LUT) of the respective optimal blot.)

For both experiments, you can see that saving the blot to image the next day is not a very good choice. This is because the secondary antibody and/or the chemiluminescent Western blot substrate is not stable enough for acceptable photon emission when digitally images after the day it is applied.

Table 1 Optimal Blot Unsatisfactory Blot Unsatisfactory Blot
Images Optimal Chemiluminescent Western Blot Unsatisfactory Chemiluminescent Western Blot Unsatisfactory Chemiluminescent Western Blot
Conditions:
Substrate SuperSignal® West Dura1 SuperSignal West Dura1 SuperSignal West Dura1
Processing Time Same Day Next Day Next Day
Detection Process HRP secondary incubated, washed, and substrate added immediately before imaging. HRP secondary incubated, washed, and substrate added day before imaging. HRP secondary incubated, washed, and substrate added day before imaging, then re-incubated with HRP secondary and substrate added immediately before imaging.
Storage Conditions Blot stored overnight dry, at room temperature Blot stored overnight dry, at room temperature
Performance LOD – 640 ng LOD – None detected LOD – 1.25 μg
Table 2 Optimal Blot Unsatisfactory Blot Unsatisfactory Blot
Images Optimal Chemiluminescent Western Blot Unsatisfactory Optimal Chemiluminescent Western Blot Unsatisfactory Optimal Chemiluminescent Western Blot
Conditions:
Substrate SuperSignal® West Dura1 SuperSignal West Dura1 SuperSignal West Dura1
Process Time Same day Next day Next day
Detection Process HRP secondary incubated, washed, and substrate added immediately before imaging. HRP secondary incubated, washed, and substrate added day before imaging. HRP secondary incubated, washed, and substrate added day before imaging, then re-incubated with HRP secondary and substrate added immediately before imaging.
Storage Conditions Blot stored overnight wet in PBS, at room temperature Blot stored overnight wet in PBS, at room temperature
Performance LOD – 640 ng LOD – None detected LOD – 1.25 μg

1SuperSignal West Dura results are comparable to those obtained with WesternSure® PREMIUM Chemiluminescent Substrate.

For more hints and tips, stay tuned to future blog posts. And if you would like to try some FREE Western Blot Analysis Software, download Image Studio™ Lite today!

Related posts:

What if Film Was No Longer Available? How Would You Capture Your Western Blot Images?

Photographic FilmFilm has been the dominant technology for capturing images for photographers, medical practitioners, and researchers for more than 250 years. Now it’s no longer the sole option. Digital technologies are beginning to impact the future of film. Here’s how and why:

  1. Digital technology is being widely adopted across many different fields including photography, medicine, and scientific research.
  2. The affordability and supply of film has been threatened with the increase of raw material and production costs.
  3. New rules and regulations have been passed in relation to global preservation and green movements.

Because of this, several prominent companies including Kodak and Fujifilm have reevaluated their business initiatives and made decisions regarding the manufacture of certain film-related products.

Get out of the DarkroomIn addition, many universities and institutions are reconsidering their rules and regulations for the disposal and use of hazardous wastes. In general, policies are being made more stringent and punishments for non-compliance more severe. In fact, many new research and medical buildings are being built without darkrooms or the equipment necessary to process film.

Being aware of how these issues, and others, affect the future of film is essential to being able to continue the same quality, or better quality work than you are producing now. Preparing for the future by considering alternative imaging options is becoming more and more essential—especially when processing film comes with additional expenditures and concerns, and requires protocols that rely on toxic chemicals and large amounts of water.

Our next blog post will show you how the cost of raw materials influences the availability and cost of film.

Related Posts:

  • What is the Future of Film Use for Western Blot Imaging?
  • The History of Film. What Does It Tell Us About The Future of Using Film for Western Blot Imaging?
  • Is Trying to Get into the Darkroom to Develop Your Western Blot Film Giving You Nightmares?

    See your darkroom nightmares come to life in this short feature presentation from LI-COR. You never know what could be behind your darkroom door . . .

    If you want to avoid future nightmares, check out the C-DiGit® Chemiluminescent Western Blot Scanner.

    Everything you love about film, without the hassles – or the darkroom!!

    Multiplex Western Blotting System Turbo-Charges Western Blot Results Output

    Example of Multiplex Western Blotting using the MPX Blotting SystemMultiplex Western blotting is a powerful tool that allows you to get more out of your Western blots. Multiplex detection becomes possible when you utilize the MPX™ (Multiplex) Blotting System and LI-COR IRDye® near-infrared fluorescent dye-labeled secondary antibodies.

    Multiplex Westerns can be imaged on any of the Odyssey® Imagers and provide results for a possible maximum of 48 targets on a single membrane — 24 per channel with two-color detection — and the option for quantitative analysis, saving you time and reagents! The MPX Blotting System can be used if you need to optimize:

    • Primary antibodies – to determine the primary antibody that has the right specificity and the right dilution for use
    • Antibody incubation times
    • Blocking conditions – which blocking buffer will give you the optimum results
    • Secondary antibodies – what dilutions is best to use without getting a lot of non-specific binding?
    • Or just about anything else you need to optimize!

    Watch this 4 minute video on how easy it is to get the most out of multiplexing with the MPX Blotting System. You can also download the handy MPX Blotter User Guide.

    WesternSure® Chemiluminescent Western Blotting Reagents from LI-COR®

    WesternSure Chemiluminescent Western Blotting ReagentsDetect your Western Blots with Confidence! Use NEW! WesternSure® Chemiluminescent Reagents from LI-COR!

    Now, in addition the great imaging systems for chemiluminescent Western blots, LI-COR Biosciences offers chemiluminescent Western blotting substrates and HRP-conjugated secondary antibodies for use in performing your BEST chemiluminescent Western blots ever. And, the WesternSure Pen is used to annotate visible protein ladders prior to chemiluminescent Western blot detection.

    WesternSure chemiluminescent Western blotting reagents offer the best performance available when compared to other competitive products on the market. WesternSure PREMIUM Chemiluminescent Substrate is a highly sensitive enhanced substrate for detecting horseradish peroxidase (HRP) on immunoblots.

    WesternSure HRP-conjugated secondary antibodies (Goat anti-Mouse and Goat anti-Rabbit) are compatible with a variety of chemiluminescent substrates and are optimized for use with WesternSure PREMIUM chemiluminescent substrates.

    Happy Blotting!

    No Darkroom? No Problem! Get the LI-COR® C-DiGit® Western Blot Scanner !

    C-DiGit Blot Scanner - a Compact Personal Chemiluminescent Imaging System
    Are you doing chemiluminescent Western blots? Have you ever found yourself with a blot that is ready to image, but the darkroom is busy or the developer is broken?

    FINALLY, image at your convenience. Keep your C-DiGit Blot Scanner on your lab bench, at your desk, or anywhere you choose (look left and see just how small and portable the C-DiGit Western Blot Scanner really is!). It can truly be YOUR personal chemiluminescent Western imager!

    The C-DiGit Chemiluminescent Western Blot Scanner maintains the simplicity of film exposures without the mess of the darkroom. You perform all of the same steps, without buying film and spending time in the darkroom. The C-DiGit Scanner gives you a complete digital replacement for film – keeping the advantages of film and eliminating many of the drawbacks – saving you time AND money!

    Watch this short video and then visit our website to get your very own C-DiGit Blot Scanner!

    NEW! IRDye® Goat Anti-Mouse IgM Secondary Antibodies from LI-COR®!

    IRDye Dye-labeled Goat anti-Mouse AntibodiesOur IRDye secondary antibody line is growing! We have recently added IRDye Goat anti-Mouse IgM (μ chain specific) secondaries labeled with:

    • IRDye 800CW (PN 926-32280)
    • IRDye 680RD (PN 926-68180) or
    • IRDye 680LT (PN 926-68080).

    Just like all of the LI-COR IRDye secondary antibodies, these are highly cross-adsorbed secondary antibody conjugates suitable for a variety of applications (see the table below).

    IRDye 800CW secondary antibodies are the antibodies of choice for a wide variety of applications in the 800 nm channel (see the list below). IRDye 800CW secondary antibodies can be used for 2-color detection when multiplexed with IRDye 680RD or IRDye 680LT secondary antibodies.

    IRDye 680RD secondary antibodies are the antibodies of choice for In-Cell Western Assay and Western blot applications in the 700 nm channel. These antibodies can be used for 2-color detection when multiplexed with IRDye 800CW secondary antibodies. These antibodies are our most universal use 700 nm channel antibodies. Start using IRDye 680RD first over other 700 nm dyes. Dilution working range 1:10,000 – 1:40,000.

    IRDye 680LT secondary antibodies have been proven the brightest signal for Western blot detection in the 700 nm channel and are comparable to Alexa Fluor 680 secondary antibodies. Choose IRDye 680LT secondary antibodies to get high signal and for specific uses of detection in the 700nm channel. These antibodies are not recommended when getting up and running on system. Once established near-infrared protocols are optimized with IRDye 680RD, IRDye 680LT can be used to optimize signals in the 700 channel. Dilution range 1:20,000 – 1:40,000. Note: optimization may be required with IRDye 680LT.

    Application IRDye 800CW
    Secondaries
    IRDye 680RD
    Secondaries
    IRDye 680LT
    Secondaries
    Western Blot
    In-Cell Western™ Assay Not Recommended
    On-Cell Western Assay Not Recommended
    Protein Array
    Immunohistochemistry
    Microscopy
    2D Gel Detection
    Tissue Section Imaging
    Small Animal Imaging Not Recommended
    Virus Titration Assay Not Known Not Known
    FRET-based Assay Not Known Not Known


    Note: Now, as of December 15, 2014, you can also get 0.1 mg sizes of all of our IRDye dye-labeled secondary antibodies. Check out our complete listing here and our new filtering tool!

    Reprobe Fluorescent Westerns with NewBlot™ Western Blot Stripping Buffers

    NewBlot Stripping Buffer IconNewBlot Western Blot Stripping Buffers are specially formulated for use with IRDye® infrared dyes (680RD, 680LT, and 800CW only) and the Odyssey® Infrared Imaging Systems to help you save time and money on recreating samples. NewBlot Stripping Buffer allows you to reuse the same blot by stripping and reprobing up to two fluorescent antibodies simultaneously.

    So, you may ask, what’s so great about NewBlot Stripping Buffer?

    • Effectively removes antibodies, yet gentle enough to retain immobilized proteins
    • Strips blots at room temperature in 20 minutes or less without an unpleasant odor
    • Allows you to reuse the same blot up to 3X! (see the data below!)
    • Offers qualitative analysis after stripping

    In the example below, beta tubulin and ERK2 were run on a gel and transferred to Immobilon®-FL PVDF membrane. They were probed with primary antibodies rabbit anti-beta-tubulin and mouse anti-ERK2 and then with IRDye 680 Goat anti-Rabbit (red) and IRDye 800CW Goat anti-Mouse (green), respectively. NewBlot PVDF Stripping Buffer was used to strip the blot, which was then reprobed with the fluorescent secondary antibodies. This was repeated 2 more times. As you can see from the series of images, there is very little apparent loss of signal in either channel in the third blot as compared to the original blot.
    Example of using NewBlot Stripping Buffer on PVDF Membranes

    NewBlot is available in two ‘flavors’: one for stripping nitrocellulose membranes and the other for stripping PVDF membranes.

    Get more power out of your blot with NewBlot Western Blot Stripping Buffers!

    Note: On August 25, 2014, we launched two new Western blot stripping buffers: NewBlot™ IR Stripping Buffer for infrared Western blots on either PVDF OR nitrocellulose membranes; and, WesternSure® ECL Stripping Buffer for chemiluminescent Western blot stripping and reprobing. BOTH do not require hazardous shipping charges, unlike many other Western stripping buffers.

    Which Western Blot Detection Method Should You Use?

    Western blots can be detected with fluorescent, chemiluminescent, or colorimetric methods. Which Western blot detection method should you choose? Find out how the three common Western blot detection methods compare to each other in terms of time, sensitivity, and other important factors. The, choose what works best for your research.

    Fluorescent detection: Fluorescent detection uses secondary antibodies labeled with fluorescent dyes, rather than enzymes. No substrates are needed.

    Enzymatic detection: Chemiluminescent and colorimetric methods use secondary antibodies labeled with enzyme reporters such as horseradish peroxidase (HRP). Signal-generating substrates are used.

    Fluorescent detection uses NIR fluorescent dyes to generate a signal.
    Secondary antibodies are labeled with dyes such as IRDye 800CW or IRDye 680RD
    • Digital imaging reveals target protein signals with high sensitivity
    • Quantitative (signal is proportional to the amount of target protein present)
    • Stable fluorescent signals are stable
    Multiplex detection of multiple protein targets without stripping and re-probing

    Chemiluminescent detection
    uses the horseradish peroxidase (HRP) enzyme and a luminescent substrate.
    • Enzymatic reaction produces light that is detected by film exposure, or digital imaging with CCD camera
    • Multiple exposures typically required to capture optimal signals and avoid signal saturation
    • Very sensitive
    • Cannot be multiplexed
    • May not be quantitative

    Colorimetric detection uses the alkaline phosphatase enzyme.
    • Enzyme converts a soluble chromogenic substrate to a colored, insoluble product that precipitates onto the membrane and produces colored bands
    • Development of the blot is stopped by washing away the soluble substrate
    • Simple and cost-effective
    • Limited sensitivity

    Comparison of Chemiluminescence and Infrared Fluorescence
    Detection for Western Blotting
    Chemiluminescence IR Fluorescence
    Sensitivity +++ +++
    Linear Dynamic Range 10-50 fold >4000 fold
    Multiplex Detection No Yes
    Signal Stability Hours Months – Years
    Enzyme Conjugate HRP
    Substrate Luminol-based None Needed
    Detection/Documentation Film Exposure/Digital Imaging Digital Imaging
    Membrane Compatibility Nitrocellulose or PVDF Nitrocellulose or PVDF

    Monitor Protein Levels and Phosphorylation with Quantitative Multiplexed Western Blots

    Molecular profiling reveals diversity of stress signal transduction cascades in highly penetrant Alzheimer’s disease human skin fibroblasts.

    Mendonsa, G., et al. PLoS ONE 4(2): e4655. doi:10.1371/journal.pone.0004655

    Aberrant signal transduction is associated with Alzheimer’s disease (AD). In skin fibroblasts of AD patients, exaggerated signal transduction occurs in response to bradykinin (BK), an inflammatory neuropeptide. BK-induced PKC signaling causes stimulation of tau phosphorylation on serine residues in AD fibroblasts, but not in normal skin fibroblasts. Quantitative Western blotting with multiplex fluorescent detection (Odyssey Imager; LI-COR Biosciences) was used to monitor protein levels and phosphorylation.

    To explore the roles of inflammatory and oxidative stress in AD pathology, this study profiled the effects of these stresses on MAPK signaling cascades in human skin fibroblasts of familial AD patients. AD fibroblasts of different genetic origins express presenilin (PS-1 or PS-2) mutated at a variety of sites. These mutations caused diverse responses to stress induced by BK or H2O2, with unique profiles of stress-induced MAPK activation, caspase-3 cleavage, and survival pathway activation. These results indicate that AD research must consider a broad spectrum of inflammatory, oxidative, and other stress factors and intracellular signaling responses.
    Reduced ERK activation in PS-1 (M146L) Alzheimer's disease fibroblasts stimulated with bradykinin.

    Figure 1. Reduced ERK activation in PS-1 (M146L) Alzheimer’s disease fibroblasts stimulated with bradykinin (BK). These fibroblasts carry a mutation in presenilin-1 associated with aberrant signaling. Mutant and control human skin fibroblasts were treated with 250 nM BK and immunoblotted for active and total ERK. Odyssey Imager was used, and fold activation was quantified. Total ERK is shown in green, and phospho-ERK in red; overlapping signals (active ERK) are shown in yellow. ERK activation was greatly reduced in PS-1 (M146L) AD fibroblasts. Graphs show mean + S.E. *p < 0.05 and **p < 0.005; n = 4. doi:10.1371/journal.pone.0004655